Ricin is a 60-65 kDa glycoprotein derived from beans of the castor plant (Montanaro et al, 1973). It is a relatively simple toxin consisting of a ricin toxin enzymatic-A (RTA) protein and a ricin toxin lectin-B (RTB) protein linked by a disulfide bond. The RTB is responsible for binding to specific sugar residues on the target cell surface and allows internalizaition of ricin by endocytosis, whereas the RTA enzymatically inactivates the ribosome to irreversibly inhibit protein synthesis. A single molecule of RTA within the cell can completely inhibit protein synthesis, resulting in cell death. Ricin is one of the most potent toxins known for humans, with an LD50 of 1-20 mg/kg body weight when ingested and 1-20 μg/kg when inhaled or injected (Audi et al, 2005); this is 400 times more toxic than cobra venom, 1000 times more toxic than cyanide, and 4000 times more toxic than arsenic. Ricin is listed on the Centers for Disease Control and Prevention (Atlanta, USA) Category B threat list and is regarded as a high terrorist risk for civilians. Unfortunately, there is currently no therapeutic or vaccine available against ricin.
The development of therapeutics against ricin has proven elusive. Chemical inhibitors targeting ricin have been developed, but these are limited by the high amounts needed for short-term effects and their own toxicity (Burnett et al, 2005; Miller et al, 2002). Development of vaccines against ricin is ongoing, but to date such vaccines have only partially protected mice against ricin (Smallshaw et al, 2007). Of the different approaches for medical countermeasures, the development of anti-ricin antibodies appears the most promising. Much work has been done on developing antibodies, both polyclonal and monoclonal, as therapeutics against the toxin. These antibodies were directed against the toxic A-chain (blocking its destructive action to the ribosome) or the lectin B-chain (preventing it from binding to and entering the cell). (Neal et al, 2010; Foxwell B M J et al, 1985)
A sheep anti-ricin F(ab)2 was developed in the United Kingdom for research and development as well as for potential emergency use. However, large amounts, about 50-100 μg of polyclonal antibodies (pAbs) (Neal et al, 2010) or 5-100 μg of mAbs (Hewetson et al, 1993; Foxwell et al, 1985), are needed either to protect or treat a mouse from ricin poisoning within a small window of time, providing significant limitations on survival. For example, 5 μg antibody delivered by the intra-peritoneal (i.p.) route had to be given within 24 h to protect mice before 5×LD50 ricin challenge (Neal et al, 2010), while 100 μg of mAb per mouse had to be given within 30 min after 10×LD50 ricin challenge (Guo et al, 2006).
It was previously reported that mice could be immunized using increasing doses of ricin, their spleens harvested, and hybridoma created by fusing the lymphocytes with myeloma cells (Furukawa-Stoffer et al, 1999). A poisoning method was then used to select clones that survived in culture medium with ricin because these secreted sufficient amounts of anti-ricin neutralizing mAbs. The antibodies from these clones had high neutralizing activity against ricin, as judged by their binding to the toxin in an enzyme linked immunosorbent assay (ELISA) and by ricin neutralization experiments. HRF4 was identified as the best mAb.
While HRF4 showed promising activity in previous studies, there remains a need in the art for highly effective molecules for neutralization of ricin activity. Such molecules would be advantageous in the development of medical countermeasure therapy.